The engineering of three-dimensional (3D) bioartificial skeletal muscle holds promise for the treatment of a variety of muscle diseases and injuries, including muscular dystrophy, traumatic muscle damage, prolonged denervation, and cardiac infarction. To improve impaired muscle function, bioartificial skeletal muscle should survive and rapidly vascularize and innervate in vivo while containing sufficient numbers of aligned muscle fibers to generate the necessary contractile force. However, state-of-the-art engineered skeletal muscle tissues consist of only a few hundred m thick sheets or muscle bundles that generate active forces too small to be clinically used for direct repair of muscle damage. Therefore, we propose to develop a novel, reproducible tissue engineering approach to fabricate relatively large skeletal muscle tissues made of aligned and differentiated muscle fibers that generate forces comparable to those of native muscle. To achieve this goal, we will integrate expertise in 3D tissue microfabrication and muscle mechanotransduction with non-invasive imaging of tissue growth and function in vitro and vascularization in vivo. Specifically, we will: 1) fabricate porous aligned skeletal myoblast networks using a cell/hydrogel micromolding approach and by stacking multiple networks create thicker skeletal muscle constructs, 2) enhance the functional properties of the muscle constructs using optimized regimens of electromechanical stimulation, and 3) endothelialize the muscle constructs with different pore sizes to optimize for construct survival and force production after implantation in a rat dorsal skinfold chamber. The obtained knowledge and technologies developed in this proposal can be applied in the future to create other tissues with complex architecture. PUBLIC HEALTH RELEVANCE: A variety of muscular diseases and injuries, including muscular dystrophy, craniofacial defects, traumatic injury and cardiac infarction would benefit from the implantation of a functional bioartificial muscle. This proposal describes a novel tissue engineering approach to fabricate relatively large bioartificial muscle tissues made of aligned and differentiated muscle fibers with potential to be used for experimental studies and tissue replacement therapies.